Objective: Despite the antioxidant, anti-inflammatory, and anti-cellular-aging activities of urolithin A (UroA), a naturally occurring postbiotic, its high lipophilicity hampers its pharmaceutical application. To overcome this limitation and improve its stability and bioavailability, a delivery system based on submicron emulsions (S-EMs) was designed. Methods: Nineteen formulations (S-EM 1 / S-EM 19) based on biocompatible excipients were prepared by two different methodologies. S-EMs were characterized evaluating macroscopical appearance and size distribution by photon correlation spectroscopy (PCS). One selected S-EM was loaded with UroA and characterized by PCS, transmission electron microscopy (TEM), small angle x-ray scattering (SAXS) and Fourier-transform infrared spectroscopy (FT-IR). In addition, pH, Z potential and syringeability were evaluated, as well as UroA entrapment efficiency by ultrafiltration and HPLC. In vitro release was studied by dialysis, while cytotoxicity was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) viability test on primary dermal human fibroblasts. Finally, the anti-inflammatory activity of S-EM-UroA was evaluated at 3, 6, and 24 hours post-injection using the carrageenan-induced paw edema model in male C57BL/6 mice, and was compared with that of the UroA suspension and S-EM alone. Results: The preformulative study enabled to select method and composition for S-EM preparation. S-EM 18 was selected for UroA loading (SEM-UroA), being characterized by mean diameter, zeta potential, pH and syringeability suitable for intraperitoneal administration. The loading of UroA up to 0.2 mg/mL did not influence S-EM physicochemical features, while maintaining technological properties for 3 months. In vitro drug release showed a biphasic profile, 2.35-fold faster in the case of SEM-UroA with respect to the unphysiological suspension of the drug. In vitro studies revealed absence of cytotoxicity at concentrations up to 5 µM. In vivo studies, conducted as a first step in assessing the potential advantages of S-EM-UroA, demonstrated a dose-dependent anti-inflammatory effect. Specifically, S-EM-UroA at 2 mg/kg reduced paw edema at 24 h (p < 00.5; One-Way ANOVA followed by Tukey’s test for multiple comparisons), and at 4 mg/kg significantly reduced edema at all time points (p < 001), whereas the UroA suspension or S-EM had no effect on carrageenan-induced paw edema at any time point showed no effect. Conclusions: Overall, these findings underscore the potential of UroA loaded S-EM as an effective delivery system, demonstrating its superiority over simple UroA suspensions in enhancing the systemic anti-inflammatory effects of the postbiotic.
Development of a submicron emulsion-based delivery system to improve the anti-inflammatory activity of urolithin A
Esposito, Elisabetta;Dzyhovskyi, Valentyn;Santamaria, Federico;Marvelli, Lorenza;Mariani, Paolo;Benedusi, Mascia;Valacchi, Giuseppe;Ferraro, Luca
2025
Abstract
Objective: Despite the antioxidant, anti-inflammatory, and anti-cellular-aging activities of urolithin A (UroA), a naturally occurring postbiotic, its high lipophilicity hampers its pharmaceutical application. To overcome this limitation and improve its stability and bioavailability, a delivery system based on submicron emulsions (S-EMs) was designed. Methods: Nineteen formulations (S-EM 1 / S-EM 19) based on biocompatible excipients were prepared by two different methodologies. S-EMs were characterized evaluating macroscopical appearance and size distribution by photon correlation spectroscopy (PCS). One selected S-EM was loaded with UroA and characterized by PCS, transmission electron microscopy (TEM), small angle x-ray scattering (SAXS) and Fourier-transform infrared spectroscopy (FT-IR). In addition, pH, Z potential and syringeability were evaluated, as well as UroA entrapment efficiency by ultrafiltration and HPLC. In vitro release was studied by dialysis, while cytotoxicity was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) viability test on primary dermal human fibroblasts. Finally, the anti-inflammatory activity of S-EM-UroA was evaluated at 3, 6, and 24 hours post-injection using the carrageenan-induced paw edema model in male C57BL/6 mice, and was compared with that of the UroA suspension and S-EM alone. Results: The preformulative study enabled to select method and composition for S-EM preparation. S-EM 18 was selected for UroA loading (SEM-UroA), being characterized by mean diameter, zeta potential, pH and syringeability suitable for intraperitoneal administration. The loading of UroA up to 0.2 mg/mL did not influence S-EM physicochemical features, while maintaining technological properties for 3 months. In vitro drug release showed a biphasic profile, 2.35-fold faster in the case of SEM-UroA with respect to the unphysiological suspension of the drug. In vitro studies revealed absence of cytotoxicity at concentrations up to 5 µM. In vivo studies, conducted as a first step in assessing the potential advantages of S-EM-UroA, demonstrated a dose-dependent anti-inflammatory effect. Specifically, S-EM-UroA at 2 mg/kg reduced paw edema at 24 h (p < 00.5; One-Way ANOVA followed by Tukey’s test for multiple comparisons), and at 4 mg/kg significantly reduced edema at all time points (p < 001), whereas the UroA suspension or S-EM had no effect on carrageenan-induced paw edema at any time point showed no effect. Conclusions: Overall, these findings underscore the potential of UroA loaded S-EM as an effective delivery system, demonstrating its superiority over simple UroA suspensions in enhancing the systemic anti-inflammatory effects of the postbiotic.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
